Genome-wide identification and evaluation of novel internal control genes for Q-PCR based transcript normalization in wheat
Genome-wide identification and evaluation of novel internal control genes for Q-PCR based transcript normalization in wheat
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DOI：
10.1007/s11103-010-9666-8
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发表时间：
2010-10-01
影响因子：
5.1
通讯作者：
Zheng, You-Liang
中科院分区：
文献类型：
--
作者：
Long, Xiang-Yu;Wang, Ji-Rui;Zheng, You-Liang
为了使用定量PCR扩增准确地定量基因表达，使用一个或多个理想的内部对照基因来标准化待比较的样品是至关重要的。理想情况下，这些内部控制基因的表达水平应该在组织、发育阶段和环境条件之间变化尽可能小。在本研究中，32个候选基因的内部控制，从9个独立的实验，其中包括333个Affyssin基因芯片小麦基因组阵列的分析。然后通过定量实时PCR与来自不同组织、发育阶段和环境条件的cDNA样品评估所选基因的表达水平。最后，选择了15个新的内参基因，并使用NormFinder、geNorm、Pearson相关系数和两倍变化法比较了它们各自的表达谱。本研究中的新的内部控制基因与13个传统的表达稳定性进行了比较。结果表明，在所有的测试cDNA样品中，7个新的内部控制基因的表达稳定性优于传统的。在传统的内部控制基因中，延伸因子1-α表现出较强的表达稳定性，而18 S rRNA、α-微管蛋白、肌动蛋白和GAPDH基因在所测试的小麦样品范围内的表达稳定性非常差。因此，使用新的内部控制基因进行归一化，将提高基因表达分析的准确性和有效性。
To accurately quantify gene expression using quantitative PCR amplification, it is vital that one or more ideal internal control genes are used to normalize the samples to be compared. Ideally, the expression level of those internal control genes should vary as little as possible between tissues, developmental stages and environmental conditions. In this study, 32 candidate genes for internal control were obtained from the analysis of nine independent experiments which included 333 Affymetrix GeneChip Wheat Genome arrays. Expression levels of the selected genes were then evaluated by quantitative real-time PCR with cDNA samples from different tissues, stages of development and environmental conditions. Finally, fifteen novel internal control genes were selected and their respective expression profiles were compared using NormFinder, geNorm, Pearson correlation coefficients and the twofold-change method. The novel internal control genes from this study were compared with thirteen traditional ones for their expression stability. It was observed that seven of the novel internal control genes were better than the traditional ones in expression stability under all the tested cDNA samples. Among the traditional internal control genes, the elongation factor 1-alpha exhibited strong expression stability, whereas the 18S rRNA, Alpha-tubulin, Actin and GAPDH genes had very poor expression stability in the range of wheat samples tested. Therefore, the use of the novel internal control genes for normalization should improve the accuracy and validity of gene expression analysis.